Optically detectable organophosphonates

ABSTRACT

The invention relates to compounds having general formula I 
     
       
         
         
             
             
         
       
     
     in which X represents an optically detectable moiety, n is an integer with 1≦n≦20, R 1  is an unbranched or branched alkyl group having 1 to 20 carbon atoms, and R 2  is hydrogen or —CH 2 —O—R 3  group, wherein R 3  has the same meaning as R 1 . These compounds are useful as inhibitors of lipolytic enzymes and can be used as tools for analysis as well as discrimination of lipolytic enzymes in biological samples.

The present invention relates to optically detectableorganophosphonates. In particular, it relates to fluorescentorganophosphonates which are lipase inhibitors.

Lipolytic enzymes are widely used biocatalysts in research and industryto achieve chemical reactions with high regio-, and stereoselectivityyielding enantiomeric alcohols or amines (Jaerger et al., TrendsBiotech. 16, 396-403 (1998); Schmid, R. and Verger, R. D., Angew. Chem.110, 1694-1720 (1998); Schmid et al., Nature 409, 258-268 (2001);Koeller et al., Nature 409, 232-240 (2001); Klibanov, Nature 409,241-246 (2001)).

Lipases catalyze hydrolysis and synthesis of triacylglycerols. Alllipases accept esters of medium (C4) and long-chain (C16) saturatedfatty acids as substrates, mainly at sn-1 or sn-3 positions (Rangheardet al., Enzyme Microb. Technol. 14, 966-974 (1992); Kirk et al.,Biocatalysis 6, 127-134 (1992); Ransac et al., J. Biol. Chem. 265,20263-20270 (1990); Rogalska et al., Chirality 5, 24-30 (1993)).

The mechanism of action of these enzymes involves the nucleophiliccleavage of an ester bond by an activated serine which belongs to thecatalytic triad Ser-His-Asp/Glu (Cygler et al., Methods Enzymol. 284,3-27 (1997); Jaeger et al., see above; Schmid, R. and Verger, R. D., seeabove).

Lipophilic p-nitrophenyl phosphonate esters are convenient tools inlipase research, e.g., for studies of substrate-enzyme interactions onthe molecular level (Ransac et al., Methods Enzymol 186, 190-231 (1997))and functional analysis, e.g., to determine the active enzyme fractionof crude or pure protein preparations (Scholze et al., Analyt. Biochem.276, 72-80 (1999); Rotticci et al., Biochim. Biophys. Acta 1483, 132-140(2000)).

These inhibitors react with the nucleophilic serine of lipases, thusleading to the formation of covalent and equimolar lipid-proteincomplexes that are stable in aqueous and organic solutions (Rotticci etal., see above; Ransac et al., 1997, see above; Bjoerkling et al.,Biorgan. Med. Chem. 2, 697-705 (1994); Zandonella et al., Eur. J.Biochem. 262, 63-69 (1999)). Such complexes represent “open” lipaseconformations and mimic the substrate-enzyme interactions in the first(tetrahedral) transition state.

Fluorescent labels in the hydrophobic tail of the organophosphonates arenot only useful for quantitative analysis of lipases but also forstudying lipid-protein interactions in the first transition state underdifferent environment (solvent) conditions (Oskolkova, O. V. andHermetter, A., Biochim. Biophys. Acta 1597, 60-66 (2002); Zandonella etal., see above).

However, there is a need for further compounds for analytical andmechanistic studies on lipolytic enzymes.

Accordingly, it is the object of the present invention to providecompounds which are useful as inhibitors of lipolytic enzymes. Inparticular, these compounds should be useful tools for analysis as wellas discrimination of lipolytic enzymes in biological samples.

This object is achieved by compounds having general formula I

in which X represents an optically detectable moiety, n is an integerwith 1≦n≦20, R₁ is an unbranched or branched alkyl group having 1 to 20carbon atoms, and R₂ is hydrogen or —CH₂—O—R₃ group, wherein R₃ has thesame meaning as R₁.

Preferably, n is an integer with 3≦n≦11.

In a preferred embodiment, R₁ is hexyl and R₂ is —CH₂—O—R₃ group with R₃being octyl or hexadecyl.

According to a further preferred embodiment, R₁ is methyl and R₂ is—CH₂—O—R₃ group with R₃ being octyl or hexadecyl.

According to another preferred embodiment, R₁ is butyl and R₂ is—CH₂—O—R₃ group with R₃ being octyl or hexadecyl.

In another preferred embodiment, the optically detectable moiety is afluorophore, preferably a perylene, pyrene or nitrobenzoxadiazole (NBD)group.

According to a further aspect, the present invention relates to the useof the new compounds for inhibiting lipolytic enzymes. It furtherrelates to the use of the new compounds for the determination and/ordiscrimination of lipolytic enzymes in biological samples.

Di-O-alkylglycero-phosphonates have already been reported as usefulinhibitors of lipase activity (Stadler et al., Biochim. Biophys. Acta1304, 229-244 (1996); Zandonella et al., see above). However, there wasno suggestion that microbial lipases would react with fluorescentphosphonate inhibitors containing amide bonds.

It has been found that the compounds according to the present inventionare able to inhibit microbial lipases. Moreover, the compounds accordingto the present invention have been found to have different inhibitoryeffects on the activity of different lipases, thereby allowingdiscrimination of lipolytic enzymes.

The invention will now be described in more detail by way of thefollowing examples and figures, wherein

FIG. 1 illustrates synthetic routes of fluorescently labelledorganophosphonates according to the present invention;

FIG. 2 illustrates the synthesis of one-chain perylene- and NBD(nitrobenzoxadiazole)-inhibitors according to the present invention; and

FIG. 3 a to 3c show inhibition of microbial lipases from Rhizopus oryzae(ROL), Pseudomonas cepacia (PCL) and Pseudomonas species (PSL),respectively, in block diagram.

EXAMPLES

Materials and Methods:

Standard chemicals were obtained from Merck. Mercaptoethanol andp-nitrophenol were from Sigma; methyl phosphonic acid dichloride,n-hexylphosphonic acid dichloride and methyl sebacoyl chloride were fromAldrich. 1-Methylimidazole, tetrazole (3.5% solution in acetonitrile,from Sigma-Aldrich), perylene, 3-carbomethoxypropionyl chloride, andN-hydroxysuccinimide were obtained from Fluka. Succinimidyl6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoate was purchasedfrom Molecular Probes,12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoic acid fromLambda Fluoreszenztechnologie GmbH (Graz, Austria).

Activated succinimide esters of fluorescently labelled acids weresynthesized from appropriate fatty acids and N-hydroxysuccinimide in thepresence of dicyclohexylcarbodiimide by a procedure essentiallydescribed elsewhere (Lapidot et al., J. Lipid Res. 8, 142-145 (1967)).

The synthesis of 2-amino-2-deoxy-1(3)-O-trityl-3(1)-O-alkyl-sn-glycerolswas performed in our laboratory according to Bucher, Ribitsch et al.(unpublished) (however, also see Doris Ribitsch, PhD-Thesis, TechnicalUniversity of Graz, 2002).

Crude Pseudomonas species lipase was purchased from Nagase BiochemicalsLtd. (Japan), the pure lipase from Pseudomonas cepacia (PCL) wasobtained from R.D. Schmid, University of Stuttgart, Germany. Rhizopusoryzae lipase was kindly provided by F. Spener and L. Haalck, Universityof Münster, Germany.

Dichloromethane (Riedel-de Haën) was dried by refluxing with phosphoruspentoxide (Merck) and subsequently distilled. Other solvents were ofanalytical grade.

TLC was carried out on silica gel 60 F₂₅₄ aluminum sheets (0.2 mm,Merck) using following mixtures: petroleum ether/ether, 2:1, v/v (system1), chloroform/methanol/acetone, 10:0.2:0.2, v/v/v (system 2), orchloroform/methanol/acetone, 10:0.5:0.5, v/v/v (system 3) as developingsolvents. For preparative TLC purification silica gel 60 aluminum sheets(0.2 mm, Merck) without fluorescent indicator were used.

Compounds were visualized under UV-light (360 nm) and by charring at120° C. after spraying with 50% sulfuric acid. Phosphorus-containingcompounds were visualized on TLC plates by staining with phosphomolybdicacid (Dittmer & Lester, J. Lipid Res. 5, 126-127 (1964)), orquantitatively determined in solutions by the method of Broekhuyse(Biochim. Biophys. Acta 260, 449-459 (1968)).

Short column chromatography was performed on Kieselgel (silica gel) 60(230-400 mesh, Merck). ¹H-NMR spectra were recorded in deuteratedsolvents at 199.97 MHz, using a Varian Gemini 200 spectrometer. Chemicalshifts (δ) are given in ppm relative to tetramethylsilane as a standard.

Positive ion ESI mass spectra were recorded on a standard Kratoselectrospray ion source that was fitted to a Kratos Profile HV-4double-focusing magnetic sector instrument (acceleration voltage 2 kV,m/z-range 25 to 2400 Da, scan speed 10 s/Dec, resolution 1700 (10%valley)). The potentials applied to the ESI source (temperature 50° C.,countercurrent flow of nitrogen 150 mL/min) were +5.98 kV at thespraying capillary, +3.12 kV at the cylinder, and +2.57 kV at theendplate. A Harvard Apparatus 22 syringe pump was used to deliver aconstant flow (6 μL/min) of dry methanol containing CsI (250 mg/L).Solutions of the samples (50 μM) in the same solvent were injected via a100-μL sample loop. The m/z values are given for the most intense peakof any isotope distribution.

3-(3-Perylenoyl)propanoic Acid Methyl Ester

To a solution of 3-carbomethoxypropionyl chloride (51.8 μL, 0.415 mmol)in 10 mL dichloromethane, 64.7 mg (0.476 mmol) aluminum chloride wereadded in portions under stirring at 0° C. The reaction was kept at thistemperature for 1 h and then 100.0 mg (0.396 mmol) perylene were addedportionwise. The mixture was kept at 0° C. for 1 h and then overnight atroom temperature, poured on about 10 g of ice, acidified with 1-3 dropsconcentrated hydrochloric acid. The product was extracted withdichloromethane (6×50 mL), washed with water (2×10 mL). The organiclayers were dried over sodium sulfate. After evaporation, the residuewas purified by column chromatography on silica gel eluting withchloroform to give the pure product perylenoylpropanoic acid methylester (121.2 mg, 83.5%). R_(f) 0.16 (system 1). ¹H-NMR (CDCl₃) (δ, ppm):2.87 (t, 2H, J_(a,b)=J_(2,3) 6.52 Hz, 2-CH₂—COO—), 3.40 (t, 2H,J_(a,b)=J_(2,3) 6.51 Hz, 3-CH₂—CO—Ar), 3.77 (s, 3H, CH₃—OCO—), 7.47-7.65(m, 3H, Ar), 7.74 (t, 2H, J 8.63 Hz, Ar), 7.95 (d, 1H, J 8.01 Hz, Ar),8.14-8.31 (m, 3H, Ar), 8.56 (d, 1H, J 8.57 Hz, Ar).

4-(3-Perylenyl)butanoic Acid

To a suspension of 3-perylenoyl propanoic acid methyl ester (360.2 mg,0.983 mmol) in 2.0 mL diethylene glycol, 98% hydrazine hydrate (150.6μL, 2.949 mmol) and powdered potassium hydroxide (275.3 mg, 4.916 mmol)were added. The reaction mixture was heated to 140° C. for 2 hrs, thento 190° C. and kept over night, cooled to room temperature, diluted with20 mL water, acidified with 0.5 mL concentrated hydrochloric acid, theprecipitate formed was filtered, washed with water to pH 7.0, dried onair or washed with acetone, then with a mixture chloroform-methanol (8:2by vol.). The filtrate was concentrated in vacuo. The crude product wasapplied onto a silica gel column and eluted with chloroform to 20%methanol in chloroform to give perylenebutanoic acid (228.9 mg, 68.8%).R_(f) 0.66 (system 3). ¹H-NMR (CDCl₃/DMSO-d₆, 20:1) (δ, ppm): 1.92(quin., 2H, 3-CH₂), 2.38 (t, 2H, J 7.00 Hz, 2-CH₂), 3.03 (t, 2H, J 7.00Hz, 4-CH₂), 7.41 (d, 1H, J 7.82 Hz, Ar), 7.43-7.64 (m, 3H, Ar), 7.76 (d,1H, J 3.42 Hz, Ar), 7.80 (d, 1H, J 3.41 Hz, Ar), 8.00 (d, 1H, J 8.49 Hz,Ar), 8.21-8.60 (m, 4H, Ar).

7-(3-Perylenoyl)heptanoic Acid Methyl Ester

The synthesis and purification of perylenoylheptanoic acid methyl esterwas achieved as described for perylenoyl propanoic acid methyl esterstarting from sebacic acid monomethyl ester chloride and perylene. Yield73.7%. R_(f) 0.23 (system 1). ¹H-NMR (CDCl₃) (δ, ppm): 1.43 (m, 4H,4-CH₂, 5-CH₂), 1.67 (m, 2H, 3-CH₂), 1.82 (m, 2H, 6-CH₂), 2.32 (t, 2H,J_(a,b)=J_(2,3) 7.42 Hz, 2-CH₂), 3.05 (t, 2H, J_(a,b)=J_(6,7) 7.35 Hz,7-CH₂), 3.69 (s, 3H, CH₃), 7.43-7.64 (m, 3H, Ar), 7.74 (t, 2H, J 8.26Hz, Ar), 7.84 (d, 1H, J 7.92 Hz, Ar), 8.13-8.30 (m, 4H, Ar), 8.48 (d,1H, J 7.54 Hz, Ar).

8-(3-Perylenyl)octanoic Acid

The synthesis and isolation was accomplished under identical conditionsas described for perylenebutanoic acid starting from7-(3-perylenoyl)heptanoic acid methyl ester yielding 60.0%perylenyloctanoic acid. R_(f) 0.63 (system 3). ¹H-NMR (CDCl₃/CD₃OD, 9:1)(δ, ppm): 1.25-1.50 (m, 6H, 3(CH₂)), 1.59 (m, 2H, 3-CH₂), 1.72 (m, 2H,7-CH₂), 2.26 (dt, 2H, J_(a,b) 1.79 Hz, J_(2,3) 7.45 Hz, 2-CH₂), 2.97 (t,2H, J_(7,8) 7.72 Hz, 8-CH₂), 7.28 (d, 1H, J 8.0 Hz, Ar), 7.43 (m, 3H,Ar), 7.59 (d, 1H, J 3.42 Hz, Ar), 7.63 (d, 1H, J 3.42 Hz, Ar), 7.83 (d,1H, J 8.54 Hz, Ar), 8.10-8.22 (m, 4H, Ar).

2-(4-(3-Perylenyl)butanoyl)amino-2-deoxy-3-O-octyl-sn-glycerol (XII)

A) To a solution of 12.0 mg (0.027 mmol) perylenebutyric acidsuccinimide ester (I) in 3 mL THF,2-amino-2-deoxy-1-O-trityl-3-O-octyl-sn-glycerol (V) (12.2 mg, 0.027mmol) was added. The reaction mixture was kept at room temperatureovernight, evaporated under a stream of argon. The residue was appliedonto a silica gel column and eluted with chloroform. Fractionscontaining the product were collected and the solvent was removed underreduced pressure. The compound (XI) (20.8 mg) was obtained with 98.6%yield. R_(f) 0.81 (system 2). Removal of the trityl blocking group from2-perylenebutanoylamino-1-O-trityl-3-O-octyl-sn-glycerol (XI) wascarried out with boron trifluoride-methanol in absolute dichloromethaneunder procedure elaborated by Hermetter et al. (Chem. Phys. Lipids 50,57-62 (1989)). Yield 70.0%.

B) The compound (XII) was achieved by an analogous procedure describedabove starting from perylenebutanoic acid succinimide ester (I) and2-amino-2-deoxy-3-O-octyl-sn-glycerol (VI) with 91.0% yield. Theproducts obtained in both cases were identical.

2-(4-(3-Perylenyl)butanoyl)amino-2-deoxy-1-O-octyl-sn-glycerol (XIV)

The compound (XIV) was obtained from an equimolar mixture ofperylenebutanoic acid succinimide ester (I) and2-amino-2-deoxy-1-O-octyl-3-O-trityl-sn-glycerol (VII) under identicalconditions described in method (A) for (XII). Overall yield after twostages was 88.3%.

2-(8-(3-Perylenyl)octanoyl)amino-2-deoxy-1-O-hexadecyl-sn-glycerol (XVI)

This product was synthesized using the same procedure as for (XII)(procedure A) starting from peryleneoctanoic acid succinimide ester (II)and 2-amino-2-deoxy-1-O-hexadecyl-3-trityl-sn-glycerol (VIII). Yield(after two stages) 90.0%.

2-(8-(3-Perylenyl)octanoyl)amino-2-deoxy-3-O-hexadecyl-sn-glycerol(XVIII)

The compound (XVIII) was prepared in 67.9% yield by the same procedureas described for (XII) (A), starting from amino glycerol (IX) andperyleneoctanoic acid succinimidyl ester

2-(6-NBD-hexanoyl)amino-2-deoxy-3-O-octyl-sn-glycerol (XX)

The amino glycerol (XX) was obtained from NBD-hexanoic acid succinimideester (III) and 2-amino-2-deoxy-1-O-trityl-3-octyl-sn-glycerol (V) asdescribed for (XII) (A). Yield 86.0%. Alternatively, the same compound(XX) was prepared under identical conditions as for (XII) method (B) butusing succinimide ester (III) and 2-amino-2-deoxy-3-O-octyl-sn-glycerol(VI) with a 100% yield.

2-(6-NBD-dodecanoyl)amino-2-deoxy-3-O-hexadecyl-sn-glycerol (XXI)

The synthesis of the long-chain alkyl acylamino glycerol (XXI) wasachieved under identical conditions as described for the compound (XII)(method B) starting from NBD-dodecanoic acid succinimide ester (IV) and2-amino-2-deoxy-3-O-hexadecyl-sn-glycerol (X). The yield comprised98.0%.

2-(4-(3-Perylenyl)butanoyl)amino-2-deoxy-3-O-octyl-sn-glycero-1-O-hexylphosphonatep-nitrophenyl Ester (XXII)

To a solution of the glycerol derivative (XII) (20.0 mg, 0.038 mmol), 40μL triethylamine, and 3.5% tetrazole solution in acetonitrile (7.4 μL,0.004 mmol) in 2 mL dichloromethane, hexyl phosphonic acid dichloride(12.9 μL, 0.076 mmol) was added at 5° C. After 1 h when the reaction wascomplete, p-nitrophenol (10.6 mg, 0.076 mmol) was added and the reactionmixture was kept overnight at room temperature. The solvent was removedunder a stream of nitrogen. The product was isolated by preparative TLCin the enveloping system chloroform/methanol/acetone, 10:0.2:0.2 by vol.Yield 13.1 mg (43.3%). ¹H-NMR (CDCl₃) (δ, ppm): 0.87 (t, 6H, 2CH₃),1.05-2.00 (m, 22H, CH₂-alkyl), 2.13 (m, 2H, 2-CH₂-acyl), 2.30 (m, 2H,3-CH₂-acyl), 3.05 (m, 2H, CH₂—O-alkyl), 3.40 (m, 4H, CH₂-O-glycerol,4-CH₂-acyl), 4.25 (m, 3H, CH₂—O—P, CH—N), 6.18 (dd, 1H, NH), 7-29-7.40(m, 3H, Ar), 7.50 (m, 3H, Ar), 7.66 (d, 1H, J.39 Hz, Ar), 7.70 (d, 1H, J2.19 Hz, Ar), 7.89 (dd, 1H, J 3.42 Hz, J 8.06 Hz, Ar), 8.17 (m, 6H, Ar).

2-(4-(3-Perylenyl)butanoyl)amino-2-deoxy-1-0-octyl-sn-glycero-3-O-hexylphosphonatep-nitrophenyl Ester (XXVI)

To a solution of 2-perylenebutanoylamino-1-O-octyl-sn-glycerol (XIV)(8.1 mg, 0.016 mmol) and N-methylimidazole (6.0 μL, 0.070 mmol) in 2 mLdichloromethane, n-hexylphosphonic dichloride (10.8 μL, 0.063 mmol) wasadded. The reaction mixture was kept at room temperature for 3 hrs.After the reaction was complete, a mixture of p-nitrophenol (10.6 mg,0.076 mmol) and N-methylimidazole (6.0 μL, 0.070 mmol) were added. After18 hrs, the solvent was evaporated under a stream of argon. Thephosphonate (XXVI) was purified by TLC in system 2. Yield 0.7 mg (5.7%).

2-(8-(3-Perylenyl)octanoyl)amino-2-deoxy-3-O-hexadecyl-sn-glycero-1-O-hexylphosphonatep-nitrophenyl Ester (XXIII)

The phosphonate (XXIII) was prepared under identical conditions asdescribed for (XXII) with 6.3% yield and under conditions as for (XXIV)as well (yield 31.6%) starting from (XVIII).

2-(8-(3-Perylenyl)octanoyl)amino-2-deoxy-1-O-hexadecyl-sn-glycero-3-O-hexylphosphonatep-nitrophenyl Ester (XXVII)

The synthesis and isolation was accomplished according to the proceduredescribed for (XXII) starting from the amino glycerol analogue (XVI).Yield 26.1%.

2-(NBD-hexanoyl)amino-2-deoxy-3-O-octyl-sn-glycero-1-O-hexylphosphonatep-nitrophenyl Ester (XXIV)

The product (XXIV) was obtained from fluorescent labeled glycerol (XX)under identical conditions as described for (XXII) with 27.8% yield.

2-(NBD-dodecanoyl)amino-2-deoxy-3-O-hexadecyl-sn-glycero-1-O-hexylphosphonatep-nitrophenyl Ester (XXV)

The compound (XXI) was phosphorylated as described for the preparationof phosphonate (XXII) using acylamino 0-alkyl glycerol (XXI) as astarting material. The yield comprised 4.4%.

((6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)aminoethanol (XXX)

A mixture of NBD-hexanoic acid succinimidyl ester (12.5 mg, 0.032 mmol)and 2-aminoethanol (2 μL, approx. 0.032 mmol) in 2 mL THF was kept for30 min. After TLC showed the reaction complete, the solvent was removedunder a stream of argon. The residue was dissolved in achloroform/methanol/water mixture (65:25:4 by vol.) (5 mL), and 1 g of aresin Dowex 50W×8 (H⁺-form) was added. After stirring for 30 min, theproduct (XXX) dissolved was filtered and the resin was washed with thesame solvent system (3×5 mL). The filtrate was evaporated under reducedpressure. TLC analysis showed single fluorescent spot with R_(f) 0.69and no aminoethanol after spraying with ninhydrine. Yield 10.6 mg(98.3%).

O-(((6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)aminoethyl-O-(p-nitrophenyl)n-hexylphosphonate (XXXI)

Using the method described for phosphonate (XXII), NBD-labeledhexylphosphonate was obtained starting from the respective alcohol(XXX). The yield comprised 11.1%.

Determination of Lipase Activity

Inhibition of lipase activity by the water-insoluble phosphonates wasperformed in Triton X-100 micelles (2 mM final concentration) andmeasured by a continuous fluorescence method according to Duque et al,(J. Lipid Res. 37, 868-876 (1996)) using3-O-hexadecyl-2-pyrenedecanoyl-1-trinitrophenylaminododecanoyl-sn-glycerolas a substrate. Lipase activity was determined at 30° C. from thedequenching of pyrene fluorescence at 378 nm (excitation at 342 nm, slitwidths 5.0 nm each) (the initial linear increase in fluorescenceintensity after addition of the lipase was used to measure enzymeactivity). The measurements were performed in ethanol-water (1:3 byvol.) as a solvent.

Inhibition Experiments

Triton X-100 specially purified for membrane research (Hofmann La Roche)(10 μL of a 100 mM stock solution in chloroform) and the appropriatevolume of a phosphonate solution in chloroform were mixed and thesolvent was removed under a stream of nitrogen. The residue wasdispersed in an 100 μM Tris-HCl puffer (pH 7.40 at 37° C.) by vigorousvortexing followed by addition of an aliquot of a lipase solution,giving a total volume of 500 μL. Incubations were performed at 4° C. Thepercentage of inhibition after 16 hrs in the reaction mixture wasdetermined by measuring the residual lipase activity of appropriatealiquots (3 μL) as indicated above. Controls were run in parallel underidentical conditions but in the absence of inhibitors.

Spectroscopic Analyses

A molar absorbance coefficient of 22000 M⁻¹ cm⁻¹ at 466 nm in methanolor 29000 M⁻¹ cm⁻¹ at 442 nm in ethanol for phosphonate inhibitorslabelled with NBD or perylene, respectively, was used to determine theconcentration of the fluorescent phosphonates (measurements wereperformed on a Hitachi U-3210 spectrophotometer) (Haugland, Handbook offluorescent probes, Molecular Probes, p. 293 (1996)).

Results:

Chemical Syntheses

Eight novel phosphonic acid esters fluorescently labeled with perylenebutanoic, perylene octanoic, NBD-hexanoic or NBD-dodecanoic acid weresynthesized. Perylene and NBD-labels were chosen because of the longerwavelength absorption and emission as compared to pyrene-containinginhibitors, which have been synthesized and characterized previously(Zandonella et al., see above).

For the preparation of perylene labelled lipase inhibitors, thecorresponding fluorescently labelled perylene fatty acids were needed.Friedel-Crafts acylation of perylene with appropriate dicarbonic acidmonomethyl ester chlorides gave perylenoyl propionic acid methyl esterand perylenoyl heptanoic acid methyl ester. It is known that perylene isonly acylated at C-3 position under these reaction conditions (Zinke etal., Ber. B75, 1042-1048 (1940)). The ¹H-NMR spectra of the synthesizedperylenoyl propionic and perylenoyl heptanoic acids (protons of thealkyl and aromatic moieties) confirmed the indicated structures of theacylation products. Kizhner-Wolf reduction of the keto-groups and theconcomitant deblocking of the methyl protective groups gaveperylenebutanoic and peryleneoctanoic acids, respectively. Proton NMRspectra were in agreement with the ascribed structures.

The synthetic routes leading to the fluorescently labelledtriacylglycerol analogues are outlined in FIG. 1. The respectivecompounds are alkylacyl(amino)deoxyglycerophosphonic acids andalkyloxyphosphonic acid derivatives and three major crucial steps areinvolved in their synthesis that are: 1) attachment of a fluorescentacyl chain to 1(3)-alkyl-2-aminodeoxy-3(1)-trityl-sn-glycerol, 2)detritylation, and 3) phosphorylation of the resultantalkylacyl(amino)deoxyglycerois.

In principle, the acylation of an amino group can be performed usingacid anhydride (Singh et al., J. Carbohydr. Chem. 8, 199-216 (1989)),acyl-halides (Zimmerman et al., j. Carbohydr. Chem. 7, 435-452 (1988);Dijkman et al., Biochim. Biophys. Acta 1043, 67-74 (1990)), mixedcarbodiimide (Hammarstroem, J. Lipid Res. 12, 760-765 (1971)), or mixedanhydrides formed by ethylchloroformate and a fatty acid (Acquotti etal., Chem. Phys. Lipids 40, 71-86 (1986)).

These procedures have several disadvantages especially because theyrequire a large molar excess of acylating reagents and long reactiontimes. The use of acyl chlorides was not convenient in our case becauseof the possible modification of fatty acid derivatives during acylation.Activated fatty acid esters (for example, p-nitrophenyl esters) havealso successfully been applied e.g. for sphingolipid synthesis (Tkaczuket al., J. Org. Chem. 46, 4393-4398 (1981); Groenberg et al.,Biochemistry 30, 10746-10754 (1991); Kann et al., Biochemistry 30,7759-7766 (1991); Shibuya et al., Chem. Pharm. Bull. 40, 1154-1165(1992)).

However, acylation with p-nitrophenyl esters of perylene butyric andperylene octanoic acids may give only low yields of acylation productsprobably because of the close vicinity of a bulky fluorophore and theester group (Oskolkova et al., Chem. Phys. Lipids 99, 73-86 (1999)).Thus, we used N-hydroxysuccinimide esters of fluorescently labelledfatty acids to acylate selectively amino groups in the presence of freehydroxy groups with high yields (Julina et al., Helv. Chim. Acta 69,368-373 (1986)). In the present invention, this simple method gave highyields of acylation products (e.g., 91.0% yield of (XII) when2-amino-2-deoxy-3-O-octyl-sn-glycerol (VI) was acylated withperylenebutanoic acid succinimide ester).

The protective trityl group of the labelledalkylacylaminodeoxytritylglycerols was removed under standard conditionsas described for the synthesis of diradylglycerols (Hermetter etal.,/989, see above) using boron trifluoride-methanol. The obtainedfluorescent alkylacylaminodeoxyglycerols (XI-XVIII) showed fluorescencespectra typical for perylene-labelled compounds (Johanson et al., J. Am.Chem. Soc. 109, 7374-7381 (1987)).

Two further problems are encountered in the synthesis of fluorescentphosphonate inhibitors. Reaction yields of phosphorylation ofglycerolipids tend to be low. Secondly, the fluorescently labelled fattyacids and, as a consequence, the fluorescently labelled glycerolipidintermediates are expensive and this is one special reason why yieldsshould be as high as possible.

The diradylglycerols were reacted with alkylphosphonic acid dichloridein the presence of N-methylimidazole as a base followed by substitutionof the second chloro atom at phosphorus by p-nitrophenol. Although thesetwo steps were carried out consecutively in a one-pot reaction, productyields were rather low (5.7% for2-deoxy-2-(4-perylenylbutanoyl)amino-1-O-octyl-3-O-hexylphosphonate(XXVI) or 6.3% for2-deoxy-2-(8-perylenyloctanoyl)amino-3-O-hexadecyl-1-O-hexylphosphonate(XXIII)) most likely because of the close vicinity of the amido group tothe phosphoric acid moiety, which as a consequence, can lead to anintramolecular cyclic side-product (oxazaphospholane) in considerableamounts.

Such compounds can in fact be prepared from2-deoxy-2-amino-phosphocholine in the presence of phosphoroxytrichloride(Deigner et al., Chem. Phys. Lipids 61, 199-208 (1992)). To avoidformation of such compounds we used a modification of thephosphorylation procedure as described by Zhao et al. (Tetrahedron Lett.49, 363-368 (1993)) for the synthesis of phosphonate esters. Thisapproach used tetrazole as a catalyst and has already been shown to besuccessful for the synthesis of phosphonates from sterically hinderedalcohols such as menthol and testosterone (Zhao et al., see above) orfor the synthesis of organophosphonate esters (Rotticci et al., seeabove).

In our case, the yields of products were significantly improved whentetrazole was used as a catalyst (e.g., yields of2-deoxy-2-(8-peryleneoctanoyl)amino-3-O-hexadecyl-1-O-hexylphosphonate(XXIII) were 6.3% in the absence of tetrazole and 31.6% in its presence(Table 1)). For the other glycerolipid phosphonate analogues, yieldswere above 25% if synthesized by the tetrazole-catalyzed reaction.

TABLE 1 Comparison of chromatographic characteristics of fluorescentphosphonates synthesized and their yields after phosphorylation. Yieldof phosphorylation, % Chromato- with graphic tetrazole Num- behaviorwithout as Compounds ber R_(f) catalyst catalyst

XXII 0.33^(a) n.d. 43.3

XXVI 0.33^(a) 5.7 n.d.

XXIII 0.46^(a) 6.3 31.6

XXVII 0.46^(a) n.d. 26.1

XXXI 0.04^(b) n.d. 11.1

XXIV 0.16^(b) n.d. 27.8

XXV 0.55^(b) n.d. 4.4 n.d. = not detectable ^(a)System for TLC:chloroform/methanol/acetone, 10:0.2:0.2 by vol.; ^(b)Inchloroform/methanol/acetone, 10:0.5:0.5 by vol.

As a second class of lipase inhibitors, phosphonates containingsingle-chain alkoxy groups instead of glycerolipids were prepared asfollows (FIG. 2). Typically, NBD-hexanoic acid succinimide ester wasreacted with aminoethanol yielding derivative (XXX). Phosphorylation ofthe latter compound gave phosphonate (XXXI) with 11.1% yield.

The proton signals in the ¹H-NMR-spectrum of phosphonate (XXII) wereconsistent with the indicated molecular structure. Since thephosphorylation procedures for all glycerolipids in this study wereidentical, only one proton NMR spectrum for the latter compound ispresented. UV- and fluorescence spectra of all perylene-labelledorganophosphorus compounds showed identical λ_(max) which are consistentwith their assumed chemical structures.

The perylene-(XXII, XXIII, XXVI, XXVII) and NBD-phosphonates (XXIV, XXV,XXXI) had fluorescence maxima at 448 nm and 533 nm in ethanol, typicalfor NBD containing derivatives, respectively. The ratio of fluorescentlabel and phosphorus content of all phosphonates was about 1 to 1.15,additionally confirming the structures of the compounds obtained.Moreover, in ESI mass-spectra of the compounds (XXII, XXVI, XXIII,XXVII, XXXI, XXIV, XXV) only signals corresponding to their molecularstructures were observed (Table 2).

TABLE 2 Results of mass-spectrometry analysis of organophosphonates.Compound Molecular number Formula weight Ions observed m/z_(theor)m/z_(observed) XXII C₄₇H₅₇N₂O₇P 792.39 [C₄₇H₅₇N₂O₇P•Cs]⁺ 925.30 925.4XXVI C₄₇H₅₇N₂O₇P 792.39 [C₄₇H₅₇N₂O₇P•Cs]⁺ 925.30 925.3 XXIII C₅₉H₈₁N₂O₇P960.58 [C₅₉H₈₁N₂O₇P•Cs]⁺ 1093.48 1093.4 XXVII C₅₉H₈₁N₂O₇P 960.58[C₅₉H₈₁N₂O₇P•Cs]⁺ 1093.48 1093.4 XXXI C₂₆H₃₅N₆O₉P 606.22[C₂₆H₃₅N₆O₉P•Cs]⁺ 739.13 739.3 XXIV C₃₅H₅₃N₆O₁₀P 748.36[C₃₅H₅₃N₆O₁₀P•Cs]⁺ 881.26 881.3 XXV C₄₉H₈₁N₆O₁₀P 944.58[C₄₉H₈₁N₆O₁₀P•Cs]⁺ 1077.48 1077.3

Mass-spectra were recorded as described in “Materials and Methods”.

Interaction of Inhibitors with Lipolytic Enzymes

The novel compounds were tested with respect to their ability to inhibitthree selected microbial lipases with different substrate preferences.The lipase from Rhizopus oryzae was efficiently inactivated by theperylene- and NBD-inhibitors (FIG. 3 a) at 4° C. for 16 hrs in 100 μMTris-HCl puffer at inhibitor and lipase concentrations of 1.0 mM and 0.1mM, respectively. It is noteworthy that the long-chain inhibitors(XXIII) and (XXVII) were somewhat more active as compared to thecorresponding short-chain compounds (XXII) and (XXVI). The long-chainNBD-dodecanoylamino-sn-1-phosphonate (XXV) was slightly more effectivethan its short-chain counterpart (XXIV). This is in line with theassumption that lipases prefer long-chain glycerol esters as substrates,and evidently inhibitors as well. The two-chain phosphonate (XXII) was amuch more potent inhibitor of the lipase from Pseudomonas cepacia thanthe other inhibitors used (FIG. 3 b). In contrast, the Pseudomonasspecies lipase was quantitatively inactivated by all synthesizedorganophosphonates (FIG. 3 c).

Lipases may show very different steric constraints around and withintheir active site, and as a consequence, very different substrate andinhibitor preferences (Pleiss et al., Chem. Phys. Lipids 93, 67-80(1998)). The lipases ROL, PCL and PSL, which had been chosen for theinhibition experiments with organophosphonates, are typical examples forthis structural diversity among highly homologous enzymes. Accordingly,they show different patterns of reactivity not only towards lipidsubstrates, but also to structurally related inhibitors as demonstratedherein.

As an alternative to water-soluble lipase monomers used in industry,cross-linked enzyme crystals (CLECs) (Khalaf et al., J. Am. Chem. Soc.118, 5494-5495 (1996); Lalonde et al., J. Am. Chem. Soc. 117, 6845-6852(1995); Clair et al., J. Am. Chem. Soc. 114, 7314-7316 (1992)) have beenintroduced to increase enzyme activity (Zelinski et al., Angew. Chem.109, 746-748 (1997)) and stability in organic solvents (Persichetti etal., 1995). This makes them useful catalysts which are easy to separatefrom the reaction mixture and can be repeatedly used after subsequentfiltration and washing. The inhibitors according to the invention mightalso be useful to characterize the functional quality of these systemswhich is otherwise difficult to determine.

1.-7. (canceled)
 8. The use of a compound having general formula I

in which X represents an optically detectable moiety which is afluorophore, n is an integer with 1≦n≦20, R₁ is an unbranched orbranched alkyl group having 1 to 20 carbon atoms, and R₂ is hydrogen or—CH₂—O—R₃ group, wherein R₃ has the same meaning as R₁, for inhibitinglipolytic enzymes.
 9. The use of a compound having general formula I

in which X represents an optically detectable moiety which is afluorophore, n is an integer with 1≦n≦20, R₁ is an unbranched orbranched alkyl group having 1 to 20 carbon atoms, and R₂ is hydrogen or—CH₂—O—R₃ group, wherein R₃ has the same meaning as R₁, for thedetermination and/or discrimination of lipolytic enzymes in biologicalsamples.
 10. A method for inhibiting lipolytic enzymes in a biologicalsample which comprises combining a biological sample with a compoundhaving general formula I

in which X represents an optically detectable moiety which is afluorophore, n is an integer with 1≦n≦20, R₁ is an unbranched orbranched alkyl group having 1 to 20 carbon atoms, and R₂ is hydrogen or—CH₂—O—R₃ group, wherein R₃ has the same meaning as R₁.
 11. A method foranalyzing lipolytic enzymes in a biological sample which comprisescombining a biological sample with a compound having general formula I

in which X represents an optically detectable moiety which is afluorophore, n is an integer with 1≦n≦20, R₁ is an unbranched orbranched alkyl group having 1 to 20 carbon atoms, and R₂ is hydrogen or—CH—O—R₃ group, wherein R₃ has the same meaning as R₁.
 12. The methodaccording to claim 10 or 11, wherein n is an integer with 3≦n≦11. 13.The method according to claim 10 or 11, wherein R₁ is hexyl and R₂ is—CH₂—O—R₃ group with R₃ being octyl or hexadecyl.
 14. The methodaccording to claim 10 or 11, wherein R₁ is methyl and R₂ is —CH₂—O—R₃group with R₃ being octyl or hexadecyl.
 15. The method according toclaim 10 or 11, wherein R₁ is butyl and R₂ is —CH₂—O—R₃ group with R₃being octyl or hexadecyl.
 16. The method according to claim 10 or 11,wherein said fluorophore is selected from the group consisting ofperylene, pyrene or nitrobenzoxadiazole.
 17. The method according toclaim 16 wherein said fluorophore is perylene.